1. Field of the Invention
This invention lies in the field of hemocytometry and systems in general for the counting of biological cells suspended in fluids. The focus of this invention is on automated cell counting systems.
2. Description of the Prior Art
Cell counting is of interest in a variety of clinical and research procedures, including the counting of leukocytes and erythrocytes, which is of value in the diagnosis of various diseases or abnormal conditions and in the monitoring of patients that are undergoing treatment for such diseases or conditions. Cells can be counted manually by placing a known dilution of a sample between optically clear plates that are sufficiently close to each other (typically with a spacing on the order of 100 microns) to form the cells into a single layer, magnifying an area of the layer of designated dimensions to a known magnification, and counting the cells in the magnified area through a microscope. Manual cell counters often include a grid inscribed in the counting area to lessen the burden on the user. A description of such a grid and the procedure for its use is found in Qiu, J., U.S. Pat. No. 7,329,537 B2, issued Feb. 12, 2008, “Micro-Pattern Embedded Plastic Optical Film Device for Cell-Based Assays.” Regardless of how it is done, manual cell counting is tedious and highly vulnerable to user error. Counting is commonly aided by using a high dilution of the sample to lessen the number of cells in the counting area, but the accuracy of the counting declines with every decrease in the proportion of cells that are counted.
Automation of cell counting procedures has been made possible by the use of digital imaging systems. An example of such a system is ImageJ, a Java-based image processing program developed at the National Institutes of Health and reported by Collins, T. J., “ImageJ for microscopy,” BioTechniques 43 (1 Suppl.): 25-30 (July 2007). The use of ImageJ in hematology systems is reported by Gering, T. E., and C. Atkinson, “A rapid method for counting nucleated erythrocytes on stained blood smears by digital image analysis,” J. Parasitol. 90(4): 879-81 (2004). Further disclosures of automated cell counting are Chang, J. K., et al., U.S. Pat. No. 7,411,680 B2, issued Aug. 12, 2008, “Device for Counting Micro Particles,” and Chang, J. K., et al., United States Patent Application Publication No. US 2006/0223165 A1, published Oct. 5, 2006, “Device for Counting Cells and Method for Manufacturing the Same.”
Automated cell counting systems themselves contain an inherent statistical uncertainty due to what is commonly referred to as “sampling error,” which refers to the error inherent in selecting the area in which the automated counting is performed. One of the limitations of automated cell counters that are currently available is that due to the limitations of the optical components in the instruments, the area in which cells are counted is of limited size compared to the entire area occupied by the sample. Since this limits the number of cells accordingly, and the error increases with every decrease in the number of cells being counted, the typical instrument of the prior art is constructed with a long optical path or a large footprint (the surface area on a laboratory bench that the instrument consumes), or both, to achieve an acceptable level of accuracy. This presents disadvantages to the user, particularly when the instrument is to be used in a cell culture hood.